Detection of NTRK fusions by RNA-based nCounter is a feasible diagnostic methodology in a real-world scenario for non-small cell lung cancer assessment

NTRK1, 2, and 3 fusions are important therapeutic targets for NSCLC patients, but their prevalence in South American admixed populations needs to be better explored. NTRK fusion detection in small biopsies is a challenge, and distinct methodologies are used, such as RNA-based next-generation sequencing (NGS), immunohistochemistry, and RNA-based nCounter. This study aimed to evaluate the frequency and concordance of positive samples for NTRK fusions using a custom nCounter assay in a real-world scenario of a single institution in Brazil. Out of 147 NSCLC patients, 12 (8.2%) cases depicted pan-NTRK positivity by IHC. Due to the absence of biological material, RNA-based NGS and/or nCounter could be performed in six of the 12 IHC-positive cases (50%). We found one case exhibiting an NTRK1 fusion and another an NTRK3 gene fusion by both RNA-based NGS and nCounter techniques. Both NTRK fusions were detected in patients diagnosed with lung adenocarcinoma, with no history of tobacco consumption. Moreover, no concomitant EGFR, KRAS, and ALK gene alterations were detected in NTRK-positive patients. The concordance rate between IHC and RNA-based NGS was 33.4%, and between immunohistochemistry and nCounter was 40%. Our findings indicate that NTRK fusions in Brazilian NSCLC patients are relatively rare (1.3%), and RNA-based nCounter methodology is a suitable approach for NRTK fusion identification in small biopsies.

The NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1), NTRK2 (Neurotrophic Receptor Tyrosine Kinase 2), and NTRK3 (Neurotrophic Receptor Tyrosine Kinase 3) genes are members of the TRK (tropomyosin-receptor kinase) family, playing crucial roles in cell growth, proliferation, neuronal differentiation, survival, and metabolism in central nervous system cells 5 .The NTRK fusion arises as a result of genomic rearrangements (intra-chromosomal or inter-chromosomal) that juxtapose the 3′ region of the NTRK gene with the 5′ sequencing of the partner gene, leading to the aberrant expression of the gene and constitutive activation of the kinase domain 6 .Nevertheless, screening for NTRK fusions may be complex due to the diversity of both partners and breaking points locals.Larotectinib and Entrectinib are Food and Drug Administration (FDA)-approved targeted therapies that inhibit TRK fusion proteins and benefit patients with solid tumors harboring NTRK rearrangements 7 .
The frequency of NTRK fusions varies according to the tumor type, reported in 2-17% of thyroid cancers, 5-15% of salivary gland tumors, and ~ 1% of NSCLC [8][9][10][11] .Because of the low frequency and incompletely characterized partners in tumors like NSCLC, assays allowing the detection of several fusions or a two-step screening by immunohistochemistry (IHC) followed by confirmation by RNA-based next-generation sequencing (NGS) have been recommended [12][13][14][15] .However, due to the large number of driver alterations and the scarcity of tumor tissue usually available in NSCLC patients, multiplexed assays may improve NTRK fusion detection 16 .The nCounter assay is a robust semi-automatized platform, particularly for degraded biological material, such as formalinfixed paraffin-embedded (FFPE) tissue, that offers a cost-effective solution with high specificity and sensitivity for detecting NTRK and other therapy-targeted fusions, with a reduced rate of false positive and false negative when using a custom panel with multiplex capabilities [16][17][18][19] .
Here, we aimed to evaluate the frequency of NTRK fusions in a real-world scenario of a routine molecular profile of NSCLC and assess the feasibility of a nCounter custom assay for rearrangement alterations in a Brazilian single center.

Detection of NTRK fusions by RNA-based NGS and RNA-based nCounter assays
Next, we tested the 12 IHC-positive cases for NTRK fusions using two molecular methods: NGS panel Archer FusionPlex solid tumor and our custom fusion panel nCounter Elements XT (Fig. 1).Due to the absence of biological material in the FFPE biopsies, we were able to perform the NGS test on 50.0%(n = 6/12) of the positive pan-TRK (Table 2).
Out of the six samples tested by NGS, two samples were positive for the presence of NTRK fusions (EML4-Echinoderm microtubule-associated protein-like 4)-NTRK3 and (PRKAR1A-Protein Kinase CAMP-Dependent Type I Regulatory Subunit Alpha)-NTRK1), two were negative, and two were inconclusive (Table 2 and Fig. 3).Simultaneously, we performed our custom nCounter Elements XT fusion panel in 41.7% (n = 5/12) of the positive pan-TRK samples (Table 2).From five tested samples, two were positive for the presence of NTRK fusions (NTRK1 and NTRK3) detected by 3′-5′ imbalance, and three samples were negative (Fig. 4).To corroborate the 3′-5′ imbalance results, we included the two positive NTRK fusion non-lung cancer samples in Fig. 4. Since our assay does not use specific breakpoint probes for NTRK genes, the fusion partners are not reported.
We further evaluated the concordance rate between the results obtained from NGS, IHC, and nCounter assays (Table 2).Three of the six samples tested using the NGS assay were also analyzed by the nCounter assay, with a concordance rate of 100% (n = 3/3; two positive and one negative samples).When comparing the results obtained from the NGS assay with the pan-TRK IHC assay, we observed a concordance rate of 33.4% (n = 2/6; two positive samples).Similarly, when comparing the nCounter assay with the pan-TRK IHC assay, we observed a concordance rate of 40% (n = 2/5; two positive samples).Additionally, when comparing only positive pan-TRK samples with IHC stain intensity defined as 2+ or 3+ with the NGS assay and nCounter assay (Table 3), we observed a concordance rate of 40.0%(n = 2/5) and 66.7% (n = 2/3), respectively.

Characterization of NTRK-positive patients (nCounter and NGS)
Molecularly, none of the patients had other genetic alterations in the EGFR, KRAS, and ALK genes (Table 2).Both male and female patients had no history of tobacco consumption, were diagnosed with lung adenocarcinoma, and presented no weight loss prior to 6 months of diagnosis (Table 2).The female patient was diagnosed at 38 with a stage IVA disease, which had metastasized to the lung and pleura, and received carboplatin with pemetrexed and pembrolizumab as first-line treatment, followed by carboplatin with paclitaxel as second-line treatment after disease progression.The other patient was male, diagnosed at 71 with a disease staged as IIIB, and was submitted

Discussion
In the present study, we evaluated the feasibility of assessing NTRK fusions in a real-world scenario of routine molecular profiling of consecutive 147 NSCLC, using a custom fusion panel of nCounter assay from a single Brazilian Center.www.nature.com/scientificreports/ We observed the presence of NTRK fusions (NTRK1 and NTRK3) in 1.36% (n = 2/147) of patients.Previous studies reported that the frequency of NTRK fusions ranges from 0.1 to 3.3% in NSCLC patients worldwide, with fusions in NTRK1 and NTRK3 being more common than NTRK2 7,10,15,[20][21][22][23][24][25][26][27] .In Hispanic/Latin patients with lung cancer, a recent meta-analysis reported NTRK fusions in 1% of patients 20 .A real-world study reported 3.5% (n = 10/289) of samples with pan-TRK expression 27 .The authors, due to insufficient material, were able to confirm the presence of NTRK fusion (EML4-NTRK3) in only one patient by NGS, rendering an NTRK fusion frequency of 0.35% (n = 1/289) 27 .NTRK fusions are reported predominantly in patients with no smoking history and diagnosed with metastatic disease 7,27 .Likewise, our patients with NTRK fusion were never-smokers and diagnosed with advanced disease (IVA and IIIB).Molecularly, the presence of NTRK fusions in our series was mutually exclusive with other driver mutations and fusions, as previously described 7,27 .
Additionally, we evaluated the concordance rate between pan-TRK immunohistochemistry, RNA-based NGS, and our custom nCounter assay.Since the majority of the cases were routine small biopsies, and a panel of IHC markers initially diagnosed the cases, then were further evaluated for molecular alterations, namely EGFR, KRAS, ALK, and PD-L1, no more biological material with tumor content was available for molecular validation in half of the pan-TRK-positive cases.We observed that 33.4% (n = 2/6) of tested samples using NGS were positive for NTRK fusion, and 40.0%(n = 2/5) of tested samples using nCounter were positive for NTRK fusion.We observed  a concordance rate of 100% between the RNA-based NGS assay and our custom nCounter assay for NTRK fusion detection.Similarly, previous studies reported discordances between immunohistochemistry assays and more robust techniques (RNA-based NGS and nCounter) for NTRK fusion detection 15,18,28 .This may be due to methodology limitations since the pan-TRK immunohistochemistry assay detects wild-type and aberrant TRK proteins.In contrast, the RNA-based NGS and nCounter assays detect only the fusions 29 .Importantly, detecting NSCLC patients harboring NTRK fusions is critical since the patients may benefit from targeted therapies, such as Larotrectinib and Entrectinib 7,11 .However, none of our patients were treated with Larotectinib or Entrectinib.Also, NTRK fusions are associated with resistance to EGFR-TKIs (Tyrosine Kinase Inhibitors) in NSCLC patients 7 .Thus, NTRK fusions have emerged as a pivotal biomarker for NSCLC patients.
As previously reported, a positive cutoff of at least 1% of tumor cells was defined 14 .

RNA isolation
RNA isolation was performed from FFPE tumor samples, sectioned on slides with a thickness of 10μm.One slide was stained with hematoxylin and eosin (H&E) and evaluated by a pathologist for identification, sample adequacy assessment, and selection of the tumor tissue area (minimum of 60% tumor area).RNA was isolated using the RNeasy FFPE Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.Measurement of RNA quantity was done with TapeStation 4150 (Agilent Technologies).

Fusion detection by Archer FusionPlex solid tumor
Analysis of NTRK fusion was performed using the Archer FusionPlex Custom Solid Panel with Anchored Multiplex PCR (ArcherDX, Boulder, CO, USA) as previously described 34 .Briefly, the target-enriched cDNA library was prepared with the Archer FusionPlex solid tumor (ArcherDX, Boulder, CO, USA) using an amount of 100 ng of RNA as per the manufacturer's description.In short, the reverse transcription of RNA was followed by real-time quantitative PCR (Polymerase Chain Reaction) to determine the sample quality.Then, End-repair, adenylation, and universal half-functional adapter ligation of double-stranded cDNA fragments were followed by two rounds of PCR with universal primers and gene-specific primers, covering 53 target genes that rendered the library fully functional for clonal amplification and sequencing using the MiSeq (Illumina, USA).With the Archer Analysis software version 6.0 (ArcherDX, Boulder, CO, USA), the produced libraries were analyzed for relevant fusions.

Detection of NTRK fusions by nCounter Technology
Detection of NTRK1,2,3 rearrangement was performed using the nCounter Elements XT (NanoString Technologies, Seattle, WA, USA) custom fusion panel developed at Molecular Diagnostic Laboratory, Barretos Cancer Hospital.The panel was previously designed to detect ALK, RET, and ROS1 16 and was now updated to detect MET∆ex14 35 and NTRK1/2/3 fusions.The specific probes are detailed in Table 3.Briefly, 100 ng RNA was hybridized with specific probes for 21 h at 67 °C.Hybridized complexes were purified using the PrepStation (NanoString Technologies, Seattle, WA, USA) and then hybridized in the cartridge.Finally, the cartridge was scanned by the Digital Analyzer (NanoString Technologies, Seattle, WA, USA) for counting transcripts.Normalization of transcripts was performed by the nSolver Analysis Software v4.0 (NanoString Technologies, Seattle, WA, USA) using the ratio of geometric mean for each sample and arithmetic mean for all samples for positive assays controls and reference gene (housekeeper).Samples with counts lower than 300 counts for the GAPDH gene were considered inconclusive.
Detection of NTRK1,2,3 rearrangement was based on 3′/5′ probes imbalance, and no specific probes for breakpoints were used due to the large number of partners and breakpoints for the NTRK gene.The calculation of the imbalance probes was defined by the ratio between the geometric mean of 3′ probes and the average of 5′ probes, considering thresholds for positive NTRK1/2/3 rearrangement equal to 2. Two cases were included as controls: an infant-type hemispheric glioma 34 and an infantile fibrosarcoma harboring NTRK1 and NTRK3 fusions, initially detected by RNA-based NGS (Archer FusionPlex solid tumor).All analyses were performed in R environment v3.4.1.

Statistical analysis
We described categorical variables using percentages and continuous variables using the medians for statistical analysis.To assess the concordance rate between all the techniques, we calculated the percentage of samples with concordant and discordant results between the techniques.Frequencies and medians were performed using IBM SSPS Statistics Version 25 (IBM, Armonk, Nova York, USA).Graphs were created using GraphPad Prism v5.01 (GraphPad Software Inc., Boston, Massachusetts USA).

Statement of ethics
The institutional review board-Barretos Cancer Hospital IRB-approved the study protocol (CAAE 05744712.3.0000.5437)and waived written informed consent due to the study's retrospective nature.All procedures were performed following the Helsinki Declaration.

Figure 1 .
Figure 1.Flow chart of the study design.We selected 147 FFPE cases diagnosed with NSCLC at Barretos Cancer Hospital that were routinely evaluated for molecular diagnosis.

Figure 4 .
Figure 4. Representative graph of NTRK gene fusions obtained from the analyzed samples and two positive NTRK fusion controls (cutoff = 2).The y-axis represents the packing ratio between the 3′ and 5′ regions for the NTRK genes.The x-axis represents the RNA samples analyzed in the study.

Table 1 .
Clinicopathological and molecular features of NSCLC consecutively evaluated for pan-TRK (n = 147).n, number of patients; ECOG PS (Eastern Cooperative Oncology Group Performance Status); NSCLC, Non-small cell lung cancer; a prior to 6 months to diagnosis; b including the following histologies: NSCLC NOS (not otherwise specified), neuroendocrine large cell carcinoma, adenosquamous carcinoma, c according to AJCC 8th edition; d pan-TRK IHC (immunohistochemistry).

Table 3 .
35obes of the custom NSCLC gene fusion panel of Barretos Cancer Hospital in the Elements XT nCounter.aPreviouslypublishedbyNovaes et al.16.bPreviously published by Aguado et al.35.